The hepatitis B virus (HBV) X gene product trans-activates viral and cellular genes. The X protein (pX) does not bind independently to nucleic acids. The data presented here demonstrate that pX entered into a protein-protein complex with the cellular transcriptional factors CREB and ATF-2 and altered their DNA binding specificities. Although CREB and ATF-2 alone did not bind to the HBV enhancer element, a pX-CREB or pX-ATF-2 complex did bind to the HBV enhancer. Thus, the ability of pX to interact with cellular factors broadened the DNA binding specificity of these regulatory proteins and provides a mechanism for pX to participate in transcriptional regulation. This strategy of altered binding specificity may modify the repertoire of genes that can be regulated by transcriptional factors during viral infection.

Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding protein (TBP). These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex. We report here that the hepatitis B virus protein X (pX) specifically binds to TBP in vitro. While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect. Deletion Infection by human hepatitis B virus (HBV) represents a major health problem. It is estimated that over 250 million people are chronically infected worldwide. Chronic infection has been associated with hepatocellular carcinoma (1). The HBV genome encodes four genes whose transcriptional activity is controlled by at least four promoters and two enhancer elements designated I and II. The 16.5-kDa protein X (pX), encoded by the X gene, can activate gene expression from diverse viral and cellular transcriptional control elements (2-7). pX does not interact directly with cis-acting DNA elements. The biochemical mechanism responsible for pX activity in the viral life cycle remains obscure. While little is known regarding the potential interactions between pX and cellular proteins, it is clear from numerous studies that pX may function by a number of distinct mechanisms (7). We have previously shown that pX mediates transcriptional transactivation by engaging in protein-protein interactions (8). pX was shown to enhance the DNA-binding specificity of the transcription factors ATF-2 and CREB. This interaction within the HBV enhancer element I leads to transactivation through the cAMP response element (8). Several cellular genes, including interleukin 8, intercellular adhesion molecule 1, c-myc, and major histocompatibility complex class I and class II genes, have been shown to be activated by pX via distinct transcription factors (9-13). Consistent with these findings, an NF-KB sequence located in the human immunodeficiency virus long terminal repeat was shown to be the target sequence of pX transactivation (5, 6). However, there is no direct evidence for a direct interaction between pX and these factors. pX has also been shown to increase an endogenous protein kinase C activity via signal transduction pathways (14,15). A recent report demonstrated interactions of pX with the p53 tumorsuppressor gene, and it was proposed that this interaction may impede DNA repair of host genes (16). The mechanism(s) by which pX is able to activate gene expression through a variety of promoters/enhancers has been the subject of intense investigation. Of interest is the observation that the general transcriptional factor TFIID [or the TATA-binding protein (TBP)] is the target of transactivation by a large number of viral and cellular factors. These include adenovirus ElA (17), herpes simplex virus 1 VP16 (18), Epstein-Barr virus Zta (19), human cytomegalovirus IE2 (20), human T-cell lymphotropic virus 1 Tax (21), and human immunodeficiency virus Tat (22). I...

The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liverspecific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAA-CA(GTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.The human hepatitis B virus (HBV) infects hepatocytes and causes acute and chronic hepatitis. Expression of the HBV genes appears to be modulated by transcriptional control elements that display liver specificity. This is facilitated by the interaction of trans-acting cellular factors with upstream regulatory sequences. There are four genes encoded by the viral genome (Fig. 1A): S/preS, C/e, Pol, and X (1), whose transcription is controlled by four promoters (see review, ref. 1). Two enhancer elements have been identified in the HBV genome by the use of reporter genes in heterologous systems (3-5), both of which display liver specificity (3-7). The enhAncer element, termed enhancer I, is located between 966 and 1308 nt (3, 4, 7) and also has been speculated to contain the functional domains of the X gene promoter. This enhancer has been previously shown to direct its influence on the promoters of C, X, and, to a modest extent, the S ORF (8-12). Deletion of this enhancer from the genome results in a general decline of transcription from vi-al promoters (13).Analysis of DNA-protein interactions of both enhancers has revealed binding regions for several cellular transcriptional factors (6, 14-17). These protein binding sites display homology to known sequence motifs for which factors have been identified and purified. Some of these interactions have been shown to be liver specific (6,(14)(15)(16)(17). A second element, designated as enhancer II, was identified relatively recently (5) and maps approximately within the core/pregenomic promoter at nt 1645-1803 in the HBV adw2 genome.In this report, using deletion mutagenesis studies of the enhancer/X promoter complex, we have identified key elements crucial for enhancer function and those required for X promoter activity. Two regions of DNA-protein interaction were shown to be essential for enhancer activity. The transcription factor EF-C binds to one of these sites (18), and the other site binds a...

INTRODUCTION In November 2015, the Centers for Disease Control and Prevention (CDC) sent a letter to state and territorial epidemiologists, state and territorial public health laboratory directors, and state and territorial health officials. In this letter, culture-independent diagnostic tests (CIDTs) for detection of enteric pathogens were characterized as “a serious and current threat to public health surveillance, particularly for Shiga toxin-producing Escherichia coli (STEC) and Salmonella .” The document says CDC and its public health partners are approaching this issue, in part, by “reviewing regulatory authority in public health agencies to require culture isolates or specimen submission if CIDTs are used.” Large-scale foodborne outbreaks are a continuing threat to public health, and tracking these outbreaks is an important tool in shortening them and developing strategies to prevent them. It is clear that the use of CIDTs for enteric pathogen detection, including both antigen detection and multiplex nucleic acid amplification techniques, is becoming more widespread. Furthermore, some clinical microbiology laboratories will resist the mandate to require submission of culture isolates, since it will likely not improve patient outcomes but may add significant costs. Specimen submission would be less expensive and time-consuming for clinical laboratories; however, this approach would be burdensome for public health laboratories, since those laboratories would need to perform culture isolation prior to typing. Shari Shea and Kristy Kubota from the Association of Public Health Laboratories, along with state public health laboratory officials from Colorado, Missouri, Tennessee, and Utah, will explain the public health laboratories' perspective on why having access to isolates of enteric pathogens is essential for public health surveillance, detection, and tracking of outbreaks and offer potential workable solutions which will allow them to do this. Marc Couturier of ARUP Laboratories and Melissa Miller of the University of North Carolina will explain the advantages of CIDTs for enteric pathogens and discuss practical solutions for clinical microbiology laboratories to address these public health needs.